WO2018171164A1 - Camptothecin prodrug, preparation therefor and use thereof - Google Patents

Abstract

Provided is a camptothecin prodrug. The camptothecin prodrug is a prodrug formed by camptothecin or a derivative thereof and Evans blue, and the structural formula thereof is as shown in formula (I), wherein R1 is camptothecin or a derivative group thereof; R2 is one of -CH2- or -O-; R3 is one of -CH2-, (A) or (B); X is one of S or -CH2-; and n1 and n2 are the number of repeating units and both represent an integer from 0-10. The camptothecin prodrugs of the present invention have an excellent tumour cell uptake both in vitro and in vivo, and some of the prodrugs also exhibit significant cancer cell inhibitory effects. Also provided are a method for preparing the camptothecin prodrug and the use thereof in preparing a medicine for the treatment of a cancer.

Description

Camptothecin prodrug and preparation and application thereof Technical field

The invention belongs to the technical field of drug controlled release, and in particular relates to a small molecule amphiphilic drug formed by camptothecin and Evans blue, and a preparation method and application thereof.

Background technique

Cancer is one of the leading causes of morbidity and mortality worldwide. According to the World Health Organization, there were approximately 14 million new cases worldwide in 2012, and the number of new cases is expected to increase by about 70% in the next two decades. Cancer is the second leading cause of death in the world, killing 8.8 million people in 2015. Globally, nearly one-sixth of deaths are due to cancer, and about 70% of cancer deaths occur in low- and middle-income countries. Currently, chemotherapy is one of the main treatments for cancer. However, most chemotherapy drugs, such as camptothecin, paclitaxel, doxorubicin, etc., have very low solubility in water and have adverse side effects. For example, camptothecin has significant antitumor activity in preclinical studies, but fails in clinical trials due to low solubility and serious adverse side effects. Currently, the camptothecin analogs Irinotecan and Topotecan have been approved by the US Food and Drug Administration (FDA) for the treatment of colon cancer. Irinotecan shows better water solubility and reduced side effects; however, the lethality to cancer cells is also significantly impaired.

Nanomedicine has been widely studied for cancer treatment. The use of nanoparticles not only improves the water dispersibility of these drugs, but also enhances their pharmacokinetics and in vivo distribution, improving therapeutic effects and reducing side effects. Nanoparticles assembled from small molecule amphiphilic prodrugs have been extensively studied. Small molecule amphiphilic drugs, typically linked by hydrophobic chemotherapeutic drugs to hydrophilic small molecules, such as oligoethylene glycol, polypeptide sequences, or another hydrophilic drug. Small molecule amphiphilic prodrugs can be synthesized relatively easily, have a specific chemical structure, and have high drug loading capacity compared to polymer drug conjugates. However, many small molecule amphiphilic prodrugs utilize the hydroxyl group on the lactone ring of camptothecin as a linking group and form a fairly stable ester bond, which results in extremely slow release and a significant reduction in cytotoxicity against cancer cells. Furthermore, due to the high concentration of critical aggregates, drug amphiphiles may not be able to maintain their nanostructures in vivo, and thus may not benefit from the enhanced permeability and retention (EPR) effects of nanosized carriers.

Albumin is the most common protein in the blood, about 7 nanometers in size, with a long blood half-life of about 20 days. Due to the relatively large size, the EPR effect of nanostructures was first discovered by the combination of albumin and Evans Blue. Therefore, we have invented an amphiphilic small molecule prodrug that can utilize albumin as a protein carrier and rapidly release camptothecin in cells. In the present invention, the camptothecin amphiphilic small molecule prodrug maintains the nanostructure in vitro and in vivo and is capable of rapid release in cells. At present, there are no such amphiphilic small molecule prodrugs reported at home and abroad.

Summary of the invention

The primary object of the present invention is to provide a camptothecin prodrug which can be used as a small molecule amphiphilic drug, which has excellent tumor cell uptake in vivo and in vitro, and exhibits a remarkable cancer cell inhibiting effect.

Another object of the invention is to provide a process for the preparation of said camptothecin prodrug.

It is still another object of the present invention to provide the use of the camptothecin prodrug for the preparation of a medicament for the treatment of cancer.

The above object of the present invention is achieved by the following technical solutions:

First, a camptothecin prodrug is provided, which is a prodrug of camptothecin or a derivative thereof and Evans blue, and its structural formula is as shown in formula (I).

among them,

R ¹ is a camptothecin or a derivative thereof;

R ² is -CH ₂ -, or

One of them;

R ³ is -CH ₂ -,

or

One of them;

X is one of S or -CH ₂ -;

N1, n2 are the number of repeating units, all of which are integers from 0-10.

In a preferred embodiment of the invention, the camptothecin or a derivative thereof is derived from the structure represented by formula (II) or formula (III):

Formula (II) is a chemical structural formula of camptothecin, and formula (III) is a chemical structural formula of 7-ethyl-10-hydroxycamptothecin.

In a preferred embodiment of the present invention, n1 and n2 in the formula (I) are each an integer of 0 to 5; further preferably an integer of 0 to 2.

In a further preferred embodiment of the present invention, in the formula (I), R ¹ is a campobase group, and R ² is -O- or

R ³ is

Or -CH ₂ -, X is S or -CH ₂ -, n1 is an integer of 1-2, and n2 is an integer of 0-2.

In a more preferred embodiment of the present invention, the camptothecin-based prodrug is any one of the following formulas (IV), (V), (VI) or (VII):

The present invention also provides a method for preparing the camptothecin-based prodrug, which is prepared by reacting camptothecin or a derivative thereof with a raw material having a corresponding group in an organic solvent. a camptothecin or a derivative thereof, a prodrug intermediate, and finally the intermediate is further reacted with Evans blue to prepare the camptothecin prodrug; the camptothecin or a derivative thereof is preferably from camptothecin or 7-Ethyl-10-hydroxycamptothecin.

In the preparation method of the present invention, the method for preparing the camptothecin-based prodrug of the formula (IV) or (V) is described as the preparation method I, and specifically comprises the following steps:

Ia) activation of the hydroxyl group on the camptothecin lactone ring in the presence of an acylation catalyst in an organic solvent, followed by the addition of an excess of 2,2'-dithiodiethanol or 1,6-hexanediol Obtaining an intermediate alcohol;

Ib) converting the intermediate alcohol obtained in step Ia) to a carboxylic acid-terminated intermediate acid using succinic anhydride in the presence of a catalyst;

Ic) The intermediate acid obtained in the step Ib) and the Evans blue EB-NH ₂ are mixed in an organic solvent, and a reaction is stirred by adding a condensing agent to obtain a partial camptothecin-based prodrug of the present invention.

In the preparation method I of the present invention, the organic solvent described in the step Ia) may be selected from the group consisting of dichloromethane, chloroform and tetrahydrofuran. Any one of butyl, 1,4-dioxane or dimethylformamide; preferably dichloromethane.

In the preparation method I of the present invention, the acylation catalyst of the step Ia) may be selected from any one of 4-(dimethylamino)pyridine, triethylamine or N,N-diisopropylethylamine. ; 4-(dimethylamino)pyridine is preferred.

In the preparation method I of the present invention, the catalyst of the step Ib) may be selected from any one of 4-(dimethylamino)pyridine, triethylamine or N,N-diisopropylethylamine; 4-(Dimethylamino)pyridine.

In the preparation method I of the present invention, the organic solvent described in the step Ic) may be selected from any one of dimethylformamide or dimethyl sulfoxide; preferably dimethylformamide.

In the preparation method I of the present invention, the condensing agent described in the step Ic) may be selected from the group consisting of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), N, N, N', N'-tetramethyl-O-(1H-benzotriazol-1-yl)urea hexafluorophosphate (HBTU) or 2-(7-oxidized benzotriazole)-N,N,N', Any one or a mixture of two N'-tetramethyluronium hexafluorophosphates (HATU); preferably 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate.

In a preferred embodiment of the preparation method I of the present invention, the prodrug of the formula (IV) is prepared, and the synthesis route is as follows:

Specifically, the following steps are included:

In the presence of 4-(dimethylamino)pyridine, the hydroxyl group of camptothecin is activated by using triphosgene in dichloromethane, and then reacted with an excess of 2,2'-dithiodiethanol to prepare CPT-. ss-OH; then convert CPT-ss-OH to carboxylic acid-terminated CPT-ss-COOH in an organic solvent using succinic anhydride, wherein 4-(dimethylamino)pyridine needs to be added as a catalyst; finally, CPT- ss-COOH is mixed with Evans Blue EB-NH ₂ in dimethylformamide, and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and N,N- are added. The diisopropylethylamine (DIPEA) was stirred together to prepare a prodrug of the formula (IV): CPT-ss-EB.

In another preferred embodiment of the preparation method I of the present invention, a prodrug represented by the following formula (V) is prepared:

The synthetic route is as follows:

Specifically, the following steps are included:

In the presence of 4-(dimethylamino)pyridine, the hydroxyl group of camptothecin is activated by using triphosgene in dichloromethane, and then reacted with an excess of 1,6-hexanediol to prepare CPT-cc-OH. Then, CPT-cc-OH is converted into a carboxylic acid-terminated CPT-cc-COOH using an succinic anhydride in an organic solvent, wherein 4-(dimethylamino)pyridine needs to be added as a catalyst; finally, CPT-cc-COOH is added. Mixed with Evans Blue EB-NH ₂ in dimethylformamide and added 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and N,N-diisopropyl The ethylamine (DIPEA) was stirred together to prepare a prodrug of the formula (V): CPT-cc-EB.

In the preparation method of the present invention, the method for preparing the camptothecin-based prodrug of the formula (VI) or (VII) is described as the preparation method II, and specifically comprises the following steps:

IIa) In an organic solvent, a condensing agent is added to the system to form a hydroxyl group on the camptothecin lactone ring with an excess of 3,3'-dihydroporphyrin or a carboxyl group at one end of the suberic acid in the presence of an acylation catalyst. a condensation reaction to obtain a carboxyl terminated intermediate acid;

IIb) The intermediate acid obtained in the step IIa) and the Evans blue EB-NH ₂ are mixed in an organic solvent, and a reaction is stirred by adding a condensing agent to obtain a partial camptothecin-based prodrug of the present invention.

In the preparation method II of the present invention, the organic solvent described in the step IIa) may be selected from any one of dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane or dimethylformamide; preferably tetrahydrofuran. Or dichloromethane.

In the preparation method II of the present invention, the acylation catalyst described in the step IIa) may be selected from 4-(dimethylamino)pyridine, Any of triethylamine or N,N-diisopropylethylamine; preferably 4-(dimethylamino)pyridine.

In the preparation method II of the present invention, the condensing agent described in the step IIa) is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

In the preparation method II of the present invention, the organic solvent described in the step IIb) may be selected from any one of dimethylformamide or dimethyl sulfoxide; preferably dimethylformamide;

In the preparation method II of the present invention, the condensing agent described in the step IIb) may be selected from the group consisting of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), N, N, N', N'-tetramethyl-O-(1H-benzotriazol-1-yl)urea hexafluorophosphate (HBTU) or 2-(7-oxidized benzotriazole)-N,N,N', Any one or a mixture of two N'-tetramethyluronium hexafluorophosphates (HATU); preferably 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate.

In a preferred embodiment of the preparation method II of the present invention, a prodrug represented by the following formula (VI) is prepared:

The synthetic route is as follows:

Specifically, the following steps are included:

In the presence of 4-(dimethylamino)pyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl) was used as the condensation in dichloromethane. Mixture, then react with excess 3,3'-dihydroporphyrin to prepare CPT-ss-COOH-2; finally, combine CPT-ss-COOH-2 with Evans Blue EB-NH ₂ in dimethyl The formamide is mixed, and 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) are added together to prepare a formula. Prodrug (VI): CPT-ss-EB-2.

In another preferred embodiment of the preparation method II of the present invention, a prodrug of the following formula (VII) is prepared:

The synthetic route is as follows:

Specifically, the following steps are included:

In the presence of 4-(dimethylamino)pyridine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl) was used as the condensation in dichloromethane. Mixing, then reacting with excess suberic acid to prepare CPT-cc-COOH-2; finally, mixing CPT-cc-COOH-2 with Evans Blue EB-NH ₂ in dimethylformamide, and Adding 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) together to prepare a prodrug of formula (VII) : CPT-cc-EB-2.

The invention also provides the use of the camptothecin prodrug in the preparation of a medicament for treating cancer.

The camptothecin prodrug designed and synthesized by the invention is a novel drug amphiphilic prodrug which can self-assemble into nanoparticles in vitro and combine with albumin in vivo to form an amphiphilic prodrug/albumin complex. The amphiphilic drug amphiphilic prodrug of the invention is simple to prepare, has a certain chemical structure and a fixed high drug amount, and can be directly resuspended in an aqueous solution and self-assembled into a diameter of 80±16 nm. Defined nanoparticles. The formation of nanoparticles provides prodrugs with high water dispersibility and protects the drug from hydrolysis. Therefore, the amphiphilic drug amphiphilic prodrug of the present invention is stable in an aqueous solution and hardly changes within 6 days. The test proves that the camptothecin-based amphiphilic prodrug of the present invention can be efficiently endocytosed by cancer cells, and the disulfide-linked prodrugs such as CPT-ss-EB and CPT-ss-EB-2 can be used in various types. The cancer cells show strong cytotoxicity with IC ₅₀ values 10 to 27 times lower than irinotecan. In addition, disulfide bonds such as CPT-ss-EB and CPT-ss-EB-2 can rapidly cleave in the presence of glutathione and release camptothecin, thereby maintaining its anticancer activity.

In the prior art, one potential disadvantage of small molecule drug amphiphilic prodrugs compared to polymer-drug forming nanoparticles is their relatively low stability under dilute conditions in blood circulation in vivo. However, in the present invention, the CPT-ss-EB amphiphilic prodrug can be transiently converted from an 80 nm nanoparticle to a 7 nm albumin/prodrug complex by EB-albumin binding. In vivo PET imaging studies confirmed that the drug amphiphilic prodrugs of the present invention have a long blood circulation and are capable of enriching in tumors. At half hour after injection, the half-life and area under the curve (AUC) of CPT-EB were improved by 130-fold and 30-fold compared to camptothecin. Tumor accumulation of CPT-ss-EB was 40-fold higher than that of camptothecin. At the same time, the small molecule amphiphilic prodrug of the present invention has an excellent anticancer effect on colon cancer. In general, the small molecule amphiphilic prodrug prepared by the invention has remarkable innovation and strong clinical transformation ability, and opens up a new way for the development of small molecule drug delivery system.

DRAWINGS

Figure 1 is ¹ H NMR (300 MHz, CD ₂ Cl ₂ ) of CPT-ss-OH.

2 is ¹ H NMR (300 MHz, CDCl ₃ ) of CPT-ss-COOH.

Figure 3 is ¹ H NMR (300 MHz, DMSO-d ₆ ) of CPT-ss-EB.

Figure 4 is a HPLC and ESI-MS spectrum of CPT-ss-EB.

Figure 5 is a ¹ H NMR (300 MHz, CDCl ₃ ) of CPT-cc-OH.

Figure 6 is ¹ H NMR (300 MHz, CDCl ₃ ) of CPT-cc-COOH.

Figure 7 is a HPLC and ESI-MS spectrum of CPT-cc-EB.

Figure 8 is a ¹ H NMR (300 MHz, CD ₂ Cl ₂ ) of CPT-NH ₂ .

Figure 9 is a HPL spectrum of EB-NOTA-CPT.

Figure 10 is an LC-Mass spectrum of EB-NOTA-CPT.

Figure 11 is an LC-Mass spectrum of NOTA-CPT.

Figure 12 is a measurement of the critical aggregation concentration of CPT-ss-EB.

Figure 13 is a graph showing the change in hydrodynamic diameter of CPT-ss-EB.

Figure 14 is an HPLC chromatogram of CPT-ss-EB and CPT-cc-EB after stirring for 3 days at 37 degrees pH 7.4.

Figure 15 is a graph showing the Evans fluorescence change after CPT-ss-EB was added to albumin.

Figure 16 shows the blood half-life of CPT-EB and CPT.

Figure 17 is a representation of the CPT-ss-EB prodrug. (A) Photograph of CPT-ss-EB in water (left) and after red laser pass (right); (B) Hydration diameter distribution of CPT-ss-EB in PBS; (C) CPT-ss- TEM image of EB; (E) Fluorescence spectra of CPT-ss-EB and EB-COOH with or without albumin; (D) Hydration diameter distribution of CPT-ss-EB after 5 minutes of albumin addition and (F) addition of albumin 24 Hydration diameter distribution of CPT-ss-EB after hours.

Figure 18 is the release of camptothecin (CPT) in PBS with/without 10 mM glutathione (GSH) in PBS at 37 °C.

Figure 19 shows in vitro cytotoxicity of CPT-ss-EB, CPT-cc-EB, IR and CPT different cancer cells (A) HCT116, (B) U87MG, (C) 4T1, and (D) A549.

Figure 20 is a confocal microscope image showing endocytosis of (A) CPT-cc-EB and (B) CPT-ss-EB. Blue = Hoechst (nuclear staining), green = Lyso Green tracker (lysosome), red = EB from prodrug.

Figure 21 shows the results of flow cytometry analysis of CPT-ss-EB and related compounds. Left: 450 nm channel, corresponding to fluorescence from CPT; right: 660 nm channel, corresponding to fluorescence from EB.

Figure 22 is a representative whole body coronal of HCT116 tumor-bearing mice at 5 minutes, 1 hour, 3 hours, 5 hours, 7 hours, and 24 hours after intravenous injection of [64Cu]-labeled (A) CPT-EB and (B) CPT. And lateral PET images. Distribution of CPT-EB and CPT at heart (C, with blood), tumor (D) and liver (E) at different time points after tracer injection. Quantitation is based on PET images (n=3/group). (F) Mice were sacrificed 48 hours after injection and quantitative biodistribution data of their organs and tissues were collected. White arrows/circles indicate the location of the tumor. Data are expressed as mean ± standard error (n = 3 / group).

Figure 23 is a graph showing the tumor growth inhibition of HCT116, with 5 mice per group, once every 3 days, and 5 injections.

detailed description

The present invention is specifically described by the following examples, which are only used to further illustrate the present invention, and are not to be construed as limiting the scope of the present invention. Improvements and adjustments are within the scope of the invention.

Example 1: Preparation of CPT-ss-OH

A solution of 4-dimethylaminopyridine (1.05 g, 8.60 mmol) in 10 mL of dichloromethane was added dropwise to camptothecin (1.0 g, 2.87 mmol) and triphosgene (0.315 g, 1.06 mmol) with stirring. A suspension of the mixture of anhydrous dichloromethane (200 mL) was added. After stirring for 30 minutes, 2,2'-dithiodiethanol (8.60 g, 55.8 mmol) in EtOAc (EtOAc) The mixture was washed with 50 mM aqueous hydrochloric acid (2×100 mL), water (1×100 mL) and saturated brine (1×100 mL). The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. Yield: 1.05 g (69% yield). _{^{1 H NMR (300MHz, CD 2}} Cl 2) δ8.46 (s, 1H), 8.22 (d, J = 8.3Hz, 1H), 7.99 (dd, J = 8.2,1.1Hz, 1H), 7.86 (ddd, J=6.9, 6.5 Hz, 1H), 7.70 (ddd, J=8.1, 6.9, 1.2 Hz, 1H), 7.42 (s, 1H), 5.64 (d, J=18 Hz, 1H), 5.36 (m, 1H) 4.37 (t, J = 6.0 Hz, 2H), 3.93 - 3.81 (m, 2H), 3.03 - 2.82 (m, 4H), 2.19 (tdd, J = 14.0, 12.3, 7.5 Hz, 2H), 1.01 (t, J = 7.5 Hz, 3H). Figure 1 is a ¹ H NMR spectrum of CPT-ss-OH demonstrating the success of preparing the compound.

Example 2: Preparation of CPT-ss-COOH

Succinic anhydride (400 mg, 4.00 mmol), CPT-ss-OH (204 mg, 0.386 mmol) and 4-dimethylaminopyridine (19.6 mg, 0.161 mmol) prepared in Example 1 were dissolved in anhydrous dichloromethane with stirring. (200mL). The reaction mixture was stirred at room temperature overnight then washed with water (1×100 mL), EtOAc EtOAc. The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. Yield: 201 mg (83% yield). _{^{1 H NMR (300MHz, CDCl 3}} ) δ8.44 (s, 1H), 8.32 (d, J = 8.4Hz, 1H), 7.95 (d, J = 8.2Hz, 1H) (ddd, J = 8.5,6.9, 1.4 Hz, 1H), 7.69 (ddd, J=8.1, 7.0, 1.1 Hz, 1H), 7.43 (s, 1H), 5.71 (d, J = 17.3 Hz, 1H) 5.39 (d, J = 17.3 Hz, 1H) ), 5.32 (s, 2H), 4.46-4.25 (m, 4H), 3.00-2.86 (m, 4H), 2.79-2.61 m, 2H), 2.18 (m, J = 2H), 1.01 (t, J = 7.5, 3H). ESI-MS m/z calc. Figure 2 is a ¹ H NMR spectrum of CPT-ss-COOH demonstrating the success of preparing the compound.

Example 3: Preparation of CPT-ss-EB

CPT-ss-COOH (58 mg, 0.092 mmol), EB-amine (25 mg, 0.046 mmol), benzotriazol-1-yl-oxytripyrrolidinium hexafluorophosphate (PyBOP, 48 mg) prepared in Example 2. , 0.092 mmol) and N,N-diisopropylethylamine (DIPEA, 59 mg, 0.46 mmol) were combined in dimethylformamide and stirred under nitrogen for 2 days. The reaction was quenched by the addition of excess acetic acid and purified by preparative HPLC using acetonitrile and aqueous 0.2% aqueous ( gradient: 5-95% acetonitrile). The collected purified product was lyophilized and stored at -20 ° C for later use. Yield: 21 mg (40% yield). ESI-MS m/z: calcd. ^{1 H NMR (300MHz, DMSO)} δ9.34 (s, 1H), 8.70 (d, J = 3.7Hz, 1H), 8.35 (s, 1H), 8.21-8.10 (m, 3H), 8.02 (d, 9.9 Hz, 1H), 7.88 (dt, J = 8.4, 4.1 Hz, 2H), 7.73 (dd, J = 6.9, 4.1 Hz, 1H), 7.64 (dd, J = 4.0 Hz, J = 6.5 Hz, 1H), 7.50 (d, J = 5.2 Hz, 2H), 7.09 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 10 Hz, 1H), 5.56 - 5.32 (m, 4H), 4.32 (t, J = 5.9 Hz, 2H), 4.21 (t, J = 6.2 Hz, 2H), 3.55 (t, J = 6.4 Hz, 1H), 3.06 - 2.89 (m, 3H), 2.75 (dd, J = 11.9, 5.4 Hz, 1H), 2.66-2.52 (m, 6H), 2.28 (s, 3H), 2.26 (s, 3H), 2.22 - 2.13 (m, 4H), 0.92 (t, J = 7.4 Hz). Figure 3 is a ¹ H NMR spectrum of CPT-ss-EB demonstrating the success of preparing the compound. Figure 4 is a HPLC and ESI-MS spectrum of CPT-ss-EB, which also demonstrates successful preparation of the compound and has a purity of more than 99%.

Example 4: Preparation of CPT-cc-OH

A solution of 4-dimethylaminopyridine (1.68 g, 13.8 mmol) in 10 mL of dichloromethane was added dropwise to camptothecin (1.5 g, 4.31 mmol) and triphosgene (0.473 g, 1.59 mmol) with stirring. A suspension of the mixture of anhydrous dichloromethane (200 mL) was added. After stirring for 30 minutes, 1,6-hexanediol (6.64 g, 43.1 mmol) elute The mixture was washed with 50 mM aqueous hydrochloric acid (2×100 mL), water (1×100 mL) and saturated brine (1×100 mL). The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. A gradient of ethyl acetate and hexane mixture was used as the eluent. Yield: 1.48 g (70% yield). _{^{1 H NMR (300MHz, CDCl 3}} ) δ8.41 (s, 1H), 8.23 (d, J = 8.7Hz, 1H), 7.95 (dd, J = 8.2,1.1Hz, 1H), 7.85 (ddd, J = 6.9, 6.5 Hz, 1H), 7.68 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.36 (s, 1H), 5.70 (d, J = 17.3 Hz, 1H), 5.39 (d, J = 1H) ), 5.30 (s, 2H), 4.21-4.05 (m, 2H), 3.59 (dd, J = 10.1, 6.1 Hz, 2H), 2.43-2.01 (m, 2H), 1.74-1.65 (m, 3H), 1.59-1.47 (m, 2H), 1.45-1.32 (m, 4H), 1.00 (t, J = 7.5 Hz, 3H). Figure 5 is a ¹ H NMR spectrum of CPT-cc-OH demonstrating the success of preparing the compound.

Example 5: Preparation of CPT-cc-COOH

Succinic anhydride (400 mg, 4.00 mmol), CPT-cc-OH (200 mg, 0.406 mmol) and 4-dimethylaminopyridine (19.8 mg, 0.162 mmol) prepared in Example 4 were dissolved in anhydrous dichloromethane with stirring. (200mL). The reaction mixture was stirred at room temperature overnight then washed with water (1×100 mL), EtOAc EtOAc. The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. A gradient of ethyl acetate and hexane mixture was used as the eluent. Yield: 192 mg (80% yield). ESI-MS m/z (MH+). 1H NMR (300MHz, CDCl 3) δ 8.42 (s, 1H), 8.28 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 8.3 Hz, 1H), 7.85 (ddd, J = 1H), 7.68 (ddd, J=8.1, 6.9, 1.1 Hz, 1H), 7.38 (d, J = 5.1 Hz, 1H), 5.70 (d, J = 17.3 Hz, 1H), 5.39 (d, J = 17.3 Hz, 1H) ), 5.31 (s, 2H), 4.23-3.99 (m, 4H), 2.74-2.56 (m, 4H), 2.37-2.07 (m, 2H), 1.61 (ddd, J = 12.9, 6.6 Hz, 5H), 1.44-1.23 (m, 7H), 1.00 (t, J = 7.5 Hz, 3H). Figure 6 is a ¹ H NMR spectrum of CPT-cc-COOH demonstrating the success of preparing the compound.

Example 6: Preparation of CPT-cc-EB

CPT-cc-COOH (52 mg, 0.092 mmol) prepared in Example 5, EB-amine (25 mg, 0.046 mmol), benzotriazol-1-yl-oxatripyrrolidinium hexafluorophosphate (PyBOP, 48 mg) , 0.092 mmol) and N,N-diisopropylethylamine (DIPEA, 59 mg, 0.46 mmol) were combined in dimethylformamide and stirred under nitrogen for 2 days. The reaction was quenched by the addition of excess acetic acid and purified by preparative HPLC using acetonitrile and aqueous 0.2% aqueous ( gradient: 5-95% acetonitrile). The collected purified product was lyophilized and stored at -20 ° C for later use. Yield: 23 mg (45% yield). ESI-MS m/z: Calculated 1116, found 1115 (M-H). Figure 7 is a HPLC and ESI-MS spectrum of CPT-cc-EB demonstrating successful preparation of the compound and having a purity of 99% or more.

Example 7: Preparation of CPT-ss-COOH-2

3,3'-Dihydroporphyrin acid (1197 mg, 5.7 mmol), CPT (200 mg, 0.57 mmol) and 4-dimethylaminopyridine (35 mg, 0.29 mmol) were mixed in tetrahydrofuran (200 mL). The reaction mixture was stirred at room temperature overnight then washed with aq. EtOAc EtOAc EtOAc. The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. A gradient of ethyl acetate and hexane mixture was used as the eluent. Yield: 121 mg (39% yield). ^{1 H NMR (300MHz, CDCl3)} δ8.41 (s, 1H), 8.27 (d, J = 8.7Hz, 1H), 7.95 (d, J = 8.3Hz, 1H), 7.85 (m, 1H), 7.68 ( m,1H), 7.37 (d, J=5.1 Hz, 1H), 5.70 (d, J = 17.2 Hz, 1H), 5.39 (d, J = 17.2 Hz, 1H), 5.30 (s, 2H), 2.92 2.59 (m, 8H), 2.36-2.06 (m, 2H), 1.01 (t, J = 7.5 Hz, 3H).

Example 8: Preparation of CPT-ss-EB-2

CPT-ss-COOH-2 (50 mg, 0.092 mmol) prepared in Example 7, EB-amine (25 mg, 0.046 mmol), benzotriazol-1-yl-oxytripyrrolidinium hexafluorophosphate (PyBOP) , 48 mg, 0.092 mmol) and N,N-diisopropylethylamine (DIPEA, 59 mg, 0.46 mmol) were combined in dimethylformamide and stirred under nitrogen for 2 days. The reaction was quenched by the addition of excess acetic acid and purified by preparative HPLC using acetonitrile and aqueous 0.2% aqueous ( gradient: 5-95% acetonitrile). The collected purified product was lyophilized and stored at -20 ° C for later use. Yield: 11 mg (22% yield). ESI-MS m/z: calc. ^{1 H NMR (300MHz, DMSO)} δ9.35 (s, 1H), 8.71 (d, J = 3.7Hz, 1H), 8.35 (s, 1H), 8.21-8.11 (m, 3H), 8.03 (d, 9.9 Hz, 1H), 7.89 (m, 2H), 7.74 (m, 1H), 7.65 (m, 1H), 7.51 (d, J = 5.2 Hz, 2H), 7.09 (d, J = 2.3 Hz, 1H), 7.01(d,J=10.0Hz,1H),5.56-5.31(m,4H),2.98-2.52(m,8H), 2.28(s,3H), 2.25(s,3H),2.22-2.13(m, 2H), 0.91 (t, J = 7.4 Hz).

Example 9: Preparation of CPT-cc-COOH-2

Suberic acid (991 mg, 5.7 mmol), CPT (200 mg, 0.57 mmol) and 4-dimethylaminopyridine (35 mg, 0.29 mmol) were mixed in tetrahydrofuran (200 mL) with stirring. The reaction mixture was stirred at room temperature overnight then washed with aq. EtOAc EtOAc EtOAc. The organic layer was separated and dried over anhydrous sodium sulfate. The solution was concentrated by rotary evaporator and purified by flash chromatography using a pre-filled silica column. A gradient of ethyl acetate and hexane mixture was used as the eluent. Yield: 109 mg (38% yield). 1H NMR (300MHz, CDCl3) δ 8.42 (s, 1H), 8.27 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.86 (m, 1H), 7.68 (m, 1H), 7.38 (d, J = 5.1 Hz, 1H), 5.71 (d, J = 17.3 Hz, 1H), 5.38 (d, J = 17.3 Hz, 1H), 5.31 (s, 2H), 2.46-2.08 (m, 6H), 1.68-1.21 (m, 8H), 1.00 (t, J = 7.5 Hz, 3H).

Example 10: Preparation of CPT-cc-EB-2

CPT-cc-COOH-2 (46 mg, 0.092 mmol) prepared in Example 9, EB-amine (25 mg, 0.046 mmol), benzotriazol-1-yl-oxytripyrrolidinium hexafluorophosphate (PyBOP) , 48 mg, 0.092 mmol) and N,N-diisopropylethylamine (DIPEA, 59 mg, 0.46 mmol) were combined in dimethylformamide and stirred under nitrogen for 2 days. The reaction was quenched by the addition of excess acetic acid and purified by preparative HPLC using acetonitrile and aqueous 0.2% aqueous ( gradient: 5-95% acetonitrile). The collected purified product was lyophilized and stored at -20 ° C for later use. Yield: 12 mg (21% yield). ESI-MS m/z: calc. ^{1 H NMR (300MHz, DMSO)} δ9.33 (s, 1H), 8.70 (d, J = 3.7Hz, 1H), 8.34 (s, 1H), 8.20-8.10 (m, 3H), 8.01 (d, 9.9 Hz, 1H), 7.78 (m, 2H), 7.72 (m, 1H), 7.63 (m, 1H), 7.50 (d, J = 5.2 Hz, 2H), 7.08 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 10 Hz, 1H), 5.56-5.31 (m, 4H), 2.49-2.02 (m, 12H), 1.68-1.21 (m, 8H), 0.92 (t, J = 7.4 Hz).

Example 11: Preparation of CPT-NHS activated ester

CPT-cc-COOH (27 μmol) prepared in Example 5 and N-hydroxysuccinimide (27 μmol) were dissolved in 1.0 mL of anhydrous THF at -10 °C. A solution of N,N'-dicyclohexylcarbodiimide (DCC, 27 μmol) in dry tetrahydrofuran (0.5 mL) was then added dropwise. The reaction mixture was stirred at -10 °C for 1 hour and then at room temperature overnight. The white precipitate was removed by filtration and the solvent was evaporated in vacuo. The product was recrystallized from ethyl acetate to afford white crystals (yield: 52%).

Example 12: Preparation of EB-NOTA-CPT

EB-Lys-NOTA (5.0 μmol) was dissolved in 200 μL of dimethylformamide. Then, CPT-NHS activated ester (prepared in Example 11) (6.0 μmol) in 100 μL of dimethylformamide was added, followed by N,N-diisopropylethylamine (DIPEA, 25 μmol). The reaction was stirred at room temperature for 3-4 hours. The crude product was then purified using a Higgins column. A product with a chemical purity >95% was obtained. LCMS 1529.46 (M-H). Figure 9, Figure 10 is an HPLC and LC-Mass spectrum of EB-NOTA-CPT demonstrating successful preparation of the compound and having a purity of more than 95%.

Example 13: Preparation of CPT-NH ₂

To a solution of CPT-cc-COOH (3.6 equivalents) prepared in Example 5 in 1 mL of anhydrous dimethylformamide (DMF) was added 2-(7-benzotriazole)-N,N,N ', N'-Tetramethylurea hexafluorophosphate (HATU, 4.2 equivalents). Stir the solution at room temperature for 10 minutes. Then add 10 equivalents of N,N-diisopropylethylamine, then add N- A solution of tert-butoxycarbonyl-1,2-ethanediamine (1.0 eq.) in dimethylformamide. The reaction was stirred at room temperature overnight. dimethylformamide was removed using an oil vacuum pump and extracted with dichloromethane/water. the crude mixture was evaporated organic layer was then treated with trifluoroacetic acid / dichloromethane at room temperature: 1 hour (11) and the mixture was HPLC purified on a preparative column, obtained CPT-NH _2, as a yellow solid (44 % yield). Figure 8 is a ¹ H NMR spectrum of CPT-NH ₂ demonstrating success in preparing the compound.

Example 14: Preparation of NOTA-CPT

CPT-NH ₂ (5.0 μmol) was dissolved in 200 μL of dimethylformamide. Then, NOTA-NHS activated ester (6.0 μmol) in 100 μL of dimethylformamide was added, followed by N,N-diisopropylethylamine (DIPEA, 25 μmol). The reaction was stirred at room temperature for 3-4 hours. Then, it was purified using the Higgins column method. The product was >95% chemically pure. LCMS: 920.34 (M+H) using analytical HPLC and LC-MS to confirm that the product had been purified. Figure 11 is a HPLC and LC-Mass spectrum of NOTA-CPT demonstrating successful preparation of the compound and having a purity of more than 95%.

Example 15: Critical Aggregate Concentration (CAC) Measurement

芘 was used as a fluorescent probe, and CPT-ss-EB prepared in Example 3 was dispersed in water at 1.0 mg/mL, and the hydrazine content was 6.0 × 10 ^-7 mol/L. The solution was then diluted to a constant concentration of 6.0 x 10 ^-7 mol/L to various concentrations of 284 μg/mL to 0.277 μg/mL. The excitation spectra of all samples were recorded and the emission wavelength was set at 375 nm. The I ₃₃₉ /I ₃₃₅ ratio values for all solutions were determined and plotted against the concentration on the log scale (LogC). At a CPT-ss-EB concentration <1.5 μg/mL, the intensity ratio was about 0.25, indicating that the hydrazine was in a hydrophilic environment. When the concentration is >1.5 μg/mL, the intensity ratio increases significantly, eventually reaching ~0.9, which is characteristic of ruthenium in a hydrophobic environment. Figure 12 shows the measured results showing that the critical aggregation concentration value was 1.5 μg/mL in water.

Example 16: Assembly of CPT-ss-EB in water to form nanoparticles and related characterization

Lyophilized CPT-ss-EB can be directly resuspended in water or other aqueous solutions and spontaneously self-assembles into nanoparticles due to its inherent amphiphilic nature. Due to the excellent water solubility of EB and the formation of nanoparticles, CPT-ss-EB can be dispersed in an aqueous solution at a very high concentration (5 mg/mL) (see Figure 17A left). When the red laser passes through the CPT-ss-EB solution, it shows strong light scattering, indicating that the nanoparticles have formed (see Figure 17A right). We further determined the CPT-ss-EB hydration diameter by dynamic light scattering. The results show that the nanoparticles have a number average mean hydrodynamic diameter of 80 ± 16 nm (Fig. 17B). Transmission electron microscopy (TEM) showed that the CPT-ss-EB nanoparticles were spherical with a relatively uniform size of about 57 nm (Fig. 17C). The formation of stable nanoparticles not only enhances the water dispersibility of camptothecin, but also protects it from hydrolysis. As monitored by DLS within 6 days, the nanoassemblies retained hydrodynamic dimers in PBS, further indicating their stability (Figure 13). In addition, HPLC was also used to confirm the stability of the drug amphiphiles of the present invention. After three days of incubation in PBS, only traces of degradation products were observed, and neither the CPT-ss-EB prepared in Example 3 nor the CPT-cc-EB prepared in Example 6 were detected. Free CPT (see box and inverted triangle in Figure 18) Two curves). The high stability in an aqueous environment, along with the excellent ability to repeatedly freeze and resuspend, makes the pharmaceutical amphiphiles of the present invention highly attractive as a drug delivery platform.

Example 17: Assembly of CPT-ss-EB in water to form interactions between nanoparticles and albumin

In the prior art, although some small molecule drug amphiphilic prodrugs may be stable in aqueous solution, they may not retain their integrity in vivo. A key advantage of the CPT-ss-EB amphiphilic prodrugs of the present invention is that they are converted to albumin-drug complexes by binding Evans blue to albumin. The newly formed albumin/drug complex remains a nanostructure and utilizes the long blood circulation and EPR effects of albumin to preferentially accumulate drugs in the tumor. A series of studies validated the ability of CPT-ss-EB to bind to albumin. First, we found that the EB fluorescence intensity of CPT-ss-EB increased 10-fold in the presence of excess bovine serum albumin (BSA) to the same extent as unconjugated EB-COOH (see the upper two curves in Figure 17E). .

We further performed a fluorescence kinetic study of albumin binding, monitoring EB fluorescence of CPT-ss-EB at 0.2 mg/mL (corresponding to the initial mouse plasma drug concentration after drug injection) for 20 minutes, followed by albumin addition. The final concentration was 35 mg/mL (corresponding to mouse serum albumin concentration). After the addition of albumin, EB fluorescence increased by 7.3-fold and gradually increased by 8.6-fold in 4 hours. See Figure 15 for the trend.

The dynamic mean light scattering measurement showed that the number average mean hydrodynamic diameter of the CPT-ss-EB nanoassembly changed from 80 nm to 7 nm after the addition of albumin, indicating that most of the CPT-ss-EB dissociated from the larger nanostructures. Converted to albumin/CPT-ss-EB complex. In addition, the intensity-averaged hydrodynamic diameters that are very sensitive to large nanoparticles range from 121 nm for CPT-ss-EB nanoassembly to 148 nm and 7 nm after albumin addition, indicating the presence of albumin/CPT-ss-EB complexes. And a small portion of the remaining CPT-ss-EB nanoassembly. After 1 day, both the number and intensity average hydrodynamic diameters became about 7 nm. These results indicate that the CPT-ss-EB nanocomponent is fixedly converted to the albumin/CPT-ss-EB complex by albumin binding to CPT-ss-EB.

Example 18: Camptothecin release assay

The release of camptothecin from CPT-cc-EB and CPT-ss-EB was measured by dialysis. Briefly, CPT-cc-EB or CPT-ss-EB in water was diluted with phosphate buffered saline (PBS) to give a concentration of 55 μg/mL, and 0.5 mL was transferred to a pre-impregnated dialysis cassette and placed in 25 mL. Dialysis was carried out for 72 hours in phosphate buffered saline or phosphate buffered saline containing 10 mM glutathione. An aliquot (0.1 mL) was taken from the dialysate at predetermined times and 0.1 mL of fresh phosphate buffered saline was added back to the external dialysate. The increase in camptothecin concentration was monitored by high performance liquid phase (conditions: 30% acetonitrile, containing 0.1% TFA, 1 mL/min, UV detector, 250 nm).

As a result, as shown in Fig. 18, CPT-ss-EB was very stable in PBS without GSH, and only released -3% of CPT in 72 hours (block-marked curve near the abscissa in Fig. 18) . In contrast, the existence of GSH Under the condition, more than 80% of camptothecin is released rapidly within 12 hours (the curve of the origin mark at the highest point in Fig. 18). This result indicates that the disulfide bond is easily cleaved by GSH, which subsequently leads to decomposition of the carbonate bond to produce free camptothecin. These results also indicate that CPT-ss-EB is relatively stable in normal physiological conditions (eg, saline, blood, and extracellular matrix), but rapidly re-releases high toxicity in cells, particularly in cancer cells with elevated GSH levels. CPT. As expected, without a disulfide bond, CPT-cc-EB released a limited amount (<5%) of CPT in either PBS or 10 mM GSH alone.

Example 19: In vitro cytotoxicity assay

The human glioma cell line U87MG, human colon cancer cell HCT116, human lung cancer cell line A549 or mouse breast cancer 4T1 were seeded in 96-well plates. The cells were incubated at 37 ° C in a humidified atmosphere containing 5% carbon dioxide. The medium was replaced with fresh medium 24 hours after inoculation. A preparation such as camptothecin is dissolved in a solvent and diluted with a cell culture medium. For each well, 100 μL of cell culture medium with different specified drug concentrations was added. The cells were incubated for 48 hours and after this period, with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) containing 0.5 mg/mL 100 μL of medium was used to replace the medium. After the cells were incubated with the reagent for 2 hours, the medium containing unreacted MTT was carefully removed. Then, the obtained blue crystals were dissolved in 100 μL of DMSO, and the absorbance was measured at a wavelength of 570 nm in a BioTek Synergy H4 mixed reader. The measured optical density (OD) value was subtracted from the blank and cell viability was calculated based on the relative absorbance of the control untreated cells. Calculated using GraphPad Prism 5 IC ₅₀ values and statistical analysis.

We first tested the in vitro cytotoxicity of the cell line HCT116 from colon cancer (Fig. 19A). As expected, FDA approved IR showed a much higher IC50 (1.8 [mu]M) than CPT (0.087 [mu]M). CPT-cc-EB showed minimal cytotoxicity as the reactive hydroxyl group was converted to a stable ester bond with EB. The low toxicity of CPT-cc-EB also indicates that CPT-ss-EB may have low toxicity during blood circulation, with a low GSH concentration. The IC50 value of CPT-ss-EB was determined to be 0.15 [mu]M, which is similar to high potency CPT but nearly 300 times lower than CPT-cc-EB. This indicates that intracellular GSH is capable of cleaving CPT from the reducing sensitive drug amphiphile CPT-ss-EB. In addition, the cytotoxicity of CPT-ss-EB-2 was slightly lower than that of CPT-ss-EB (IC50 was 0.29 μM), but it was also significantly higher than IR. CPT-cc-EB-2 toxicity is similar to CPT-cc-EB. It is worth noting that U87MG also showed very high sensitivity to CPT-ss-EB and CPT with IC50 values of 0.31 μM and 0.12 μM, respectively (see Figure 19B). We also demonstrated that CPT-ss-EB and CPT have similar cytotoxicity to 4T1 and A549 cancer cells (see Figure 19C, Figure 19D), although all formulations are relatively less sensitive to these cells than HCT116 cells. In summary, these studies show that the camptothecin prodrugs of the present invention not only have good water solubility and stability, but also the disulfide-linked prodrugs such as CPT-ss-EB and CPT-ss-EB-2. CPT has considerable in vitro cytotoxicity and is more potent than FDA approved IR, and cleavable linkers can significantly inhibit cancer cell proliferation.

Example 20: In vitro cellular uptake

In vitro cellular uptake of camptothecin was studied using confocal laser scanning microscopy and flow cytometry. Will EB-ss-CPT and EB-cc-CPT (10 μM) was incubated with HCT116 cells for 4 hours and incubated with Lysotracker Green and 10 μg/mL Hoechst 33342 in a cell culture incubator for 0.5 hour at 37 ° C for confocal observation. The cells were then washed three times with phosphate buffered saline and then imaged on a Zeiss LSM 780 confocal microscope. Alternatively, for flow cytometry analysis, HCT116 cells were seeded in 24-well plates, and after 24 hours, EB-ss-CPT, EB-cc-CPT, camptothecin, irinotecan, and EB-amine (10 μM per Kind of drug) 4 hours. Then, the cells were separated using trypsin and washed three times with phosphate buffered saline. The fluorescence intensity of the cells was analyzed using a BD Beckman Coulter flow cytometer. EB fluorescence: excitation 532 nm, emission 660 nm; CPT fluorescence: excitation -355 nm, emission 450 nm.

Since EB is fluorescent, especially after binding to albumin, these drug amphiphilic prodrugs can be imaged directly without the need for additional dye labeling. In a confocal microscope of HCT116 cells, the nuclei were stained by Hoechst and the lysosomes were stained with LysoTracker Green. After 4 hours of incubation, CPT-ss-EB and CPT-cc-EB were localized in lysosomes and nucleus (see Figure 20A-B, where A reflects the endocytosis of CPT-cc-EB; B reflects CPT-ss- Endocytosis of EB, indicating that the drug amphiphile of the invention can be internalized by endocytic pathway. We further performed flow cytometry analysis to try to compare cellular uptake of different formulations (Figure 21). According to camptothecin fluorescence, the CPT-ss-EB and CPT-cc-EB amphiphiles of the present invention have much higher cellular uptake than the free camptothecin, irinotecan and untreated controls. (as shown in the left figure in Figure 21). Furthermore, based on EB fluorescence, the two drug prodrugs of CPT-ss-EB and CPT-cc-EB of the present invention have high cellular uptake compared to EB and control (as shown in the right panel of Figure 21). From the above experimental results, it can be concluded that the disulfide-linked prodrugs such as CPT-ss-EB of the present invention and the carbon-carbon bond-linked prodrugs such as CPT-cc-EB can be efficiently endocytosed by HCT116 cells.

Example 21: In vivo positron emission tomography (PET) imaging of HCT116 tumors.

By subcutaneous injection of 5 × 10 ⁶ th HCT116 cells were prepared in phosphate buffered saline (100 L) was treated HCT116 xenografts in nude mice: (7 weeks old, female). When the tumor size reached 500-1000 mm ³ , the mice were used for PET imaging. Mice were anesthetized with isoflurane/oxygen (2% v/v) prior to tracer injection. The anesthetized mice were intravenously injected with ⁶⁴ Cu-labeled CPT-NOTA-EB prepared in Example 12 in phosphate buffered saline (100 μL) and CPT-NOTA prepared in Example 14 (4.44-5.55 MBq/120- 150 μCi per mouse). Mice were scanned on an Inveon DPET scanner (Siemens Medical Solutions, Malvern, PA) at the indicated time points after injection. A corrected positron emission computed tomography image without attenuation or scattering is reconstructed using a 3D ordered subset expectation maximization algorithm. Image analysis using ASI Pro VMTM software. A region of interest (ROI) is drawn on any organ of interest to calculate %ID/g.

The above mice were sacrificed 48 hours after the injection. Organs and blood are collected and weighed wet. The collected organs and blood were measured for ⁶⁴ Cu radioactivity on a gamma counter (Wallac Wizard 1480, PerkinElmer) along with a series of standard solutions. Radioactivity of organs and blood was converted to calculate the percentage of injected dose (%ID) in the target organ and the percentage of injected dose per gram of tissue (%ID/g).

By PET imaging (Fig. 22), we found that CPT-EB gradually accumulated in the tumor (Fig. 22A), while CPT did not accumulate significantly (Fig. 22B). We further quantified the distribution of CPT-EB and CPT in the heart, tumor and liver. As shown in Fig. 22C, the concentration of CPT-EB gradually decreased with time, and the blood half-life was -6.5 hours (Fig. 16). In contrast, CPT concentrations decreased rapidly with a blood half-life of only ~3 minutes. The area under the curve (AUC) of CPT-EB was 221.2 ± 17.9 h * % ID / g, which was 30 times larger than the area of CPT (7.3 ± 1.2 h * % ID / g). A 130-fold increase in half-life and a 30-fold increase in AUC demonstrated that CPT-EB has a much longer blood circulation time than CPT. More importantly, the tumor uptake of CPT-EB increased rapidly from 1.18±0.24% ID/g at 5 minutes to 5.61±1.14% ID/g at 5 hours, then gradually increased to 6.33±0.22% ID/24 hours ( As shown in Figure 22D). For CPT, the opposite trend was observed: the highest concentration was 0.79 ± 0.27% ID/g at 5 minutes post injection and attenuated to near zero at 24 hours post injection (Figure 22D). The sharp drop in CPT concentration may be due to high liver absorption, which is then digested and excreted from the body (Figure 22E). In vitro quantified organ radioactivity by gamma-counting also confirmed that CPT-EB was more efficiently accumulated in tumor tissue at 4.26 ± 0.97% ID/g in tumor tissue 48 hours after injection (Fig. 22F). It is worth noting that there is a significant amount of radioactivity in the kidney, indicating that CPT-EB may be isolated from reversibly bound albumin and excreted from the body by renal clearance. These in vivo pharmacokinetics and biodistribution clearly demonstrate that the drug amphiphilic prodrugs of the invention have longer blood circulation and higher tumor enrichment.

Example 22: In vivo treatment of HCT116 tumor-bearing mice

Preparing a suspension by subcutaneous injection of 5 × 10 ⁶ th HCT116 cells in phosphate buffered saline (100 L) in the HCT116 tumor bearing nude mice (7 weeks old, female). When the tumor was established on the 10th day after tumor inoculation, the same protocol was started by intravenous injection of 100 μL of drug (CPT-ss-EB, CPT-cc-EB, IR, CPT) or phosphate buffered saline for up to 5 times every 3 days. Treated mice (calculated according to the dose of camptothecin: 3 mg/kg). Tumor volume and mouse weight were monitored every three days. Mice were euthanized when any size of the tumor exceeded 2 cm or when the mice lost more than 20% of body weight. The volume of each type of tumor was calculated using the following formula: volume = (length x width ² )/2.

As shown in Figure 23, CPT-ss-EB showed the most effective anti-tumor effect and was able to significantly delay tumor development (see the curve of the rhombic marker near the abscissa in Figure 23). The antitumor activity of CPT-cc-EB is comparable to the anti-tumor efficacy of FDA-approved irinotecan (IR) within 22 days of treatment (see the diamond-shaped marker curve and the inverted triangle marker curve at the intermediate position in Figure 23). In addition, it is important that mice treated with CPT-ss-EB and CPT-cc-EB survived well during treatment without significant weight loss; however, mice treated with camptothecin (CPT) Severe weight loss (20% reduction) and injury at the injection site occurred, and must be sacrificed on the 18th day after inoculation due to side effects (see the triangular marker curve near the abscissa in Fig. 23). These results further demonstrate that the anti-tumor effect of the camptothecin prodrugs of the present invention reaches or exceeds that of conventional conventional anti-tumor drugs, especially drug amphiphilic prodrug nanoassemblies with disulfide bonds exhibit very Effective therapeutic effects and low side effects.

Claims (10)

1. A camptothecin prodrug characterized in that it is a prodrug of camptothecin or a derivative thereof and Evans blue, and its structural formula is as in formula (I)

   

   among them,

   R ¹ is a camptothecin or a derivative thereof;

   R ² is one of -CH ₂ -, or -O-;

   R ³ is -CH ₂ -,

   

   or

   

   One of them;

   X is one of S or -CH ₂ -;

   N1, n2 are the number of repeating units, all of which are integers from 0-10.
2. The camptothecin-type prodrug according to claim 1, wherein the camptothecin or a derivative thereof is derived from one of the structures represented by the formulae (II) to (III):

   
3. The camptothecin-type prodrug according to claim 1, wherein n1 and n2 in the formula (I) are each an integer of 0 to 5; more preferably an integer of 0 to 2.
4. The camptothecin prodrug of any one of claims 1, 2 or 3, wherein in the formula (I), R ¹ is a camptothecin group, and R ² is -O- or -CH. ₂ -, R ³ is

   

   Or -CH ₂ -, X is S or -CH ₂ -, n1 is an integer of 1-2, and n2 is an integer of 0-2.
5. The camptothecin prodrug of claim 1, which has any one of the following formulas (IV), (V), (VI) or (VII):

   

   

   or

   
6. A method of preparing a camptothecin-based prodrug of formula (IV) or (V) according to claim 5, specifically comprising the steps of:

   Ia) activation of the hydroxyl group on the camptothecin lactone ring in the presence of an acylation catalyst in an organic solvent, followed by the addition of an excess of 2,2'-dithiodiethanol or 1,6-hexanediol Obtaining an intermediate alcohol;

   Ib) converting the intermediate alcohol obtained in step Ia) to a carboxylic acid-terminated intermediate acid using succinic anhydride in the presence of a catalyst;

   Ic) The intermediate acid obtained in the step Ib) and the Evans blue EB-NH2 are mixed in an organic solvent, and a reaction is stirred by adding a condensing agent to obtain the camptothecin-based prodrug.
7. The method of claim 6 wherein the organic solvent of step Ia) is selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane or dimethylformamide. Preferably, the acylation catalyst of step Ia) is selected from any one of 4-(dimethylamino)pyridine, triethylamine or N,N-diisopropylethylamine, preferably 4-( Dimethylamino)pyridine; the catalyst of step Ib) is selected from any one of 4-(dimethylamino)pyridine, triethylamine or N,N-diisopropylethylamine, preferably 4-( Dimethylamino)pyridine; the organic solvent described in step Ic) is selected from any one of dimethylformamide or dimethyl sulfoxide, preferably dimethylformamide; and the condensing agent selected in step Ic) From 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), N,N,N',N'-tetramethyl-O-(1H-benzotriazole-1- Any of urea hexafluorophosphate (HBTU) or 2-(7-oxidized benzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) Or a mixture of two, preferably 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate.
8. A method of preparing a camptothecin-based prodrug of the formula (VI) or (VII) according to claim 5, which comprises the following steps:

   IIa) In an organic solvent, a condensing agent is added to the system to form a hydroxyl group on the camptothecin lactone ring with an excess of 3,3'-dihydroporphyrin or a carboxyl group at one end of the suberic acid in the presence of an acylation catalyst. a condensation reaction to obtain a carboxyl terminated intermediate acid;

   IIb) The intermediate acid obtained in the step IIa) and the Evans blue EB-NH ₂ are mixed in an organic solvent, and a reaction is stirred by adding a condensing agent to obtain the camptothecin-based prodrug.
9. The method according to claim 8, wherein the organic solvent in the step IIa) is selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, 1,4-dioxane or dimethylformamide. Preference is given to tetrahydrofuran or dichloromethane; the acylation catalyst according to step IIa) is selected from any one of 4-(dimethylamino)pyridine, triethylamine or N,N-diisopropylethylamine, preferably 4 -(dimethylamino)pyridine; the condensing agent described in step IIa) is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the organic solvent described in step IIb) Any one selected from the group consisting of dimethylformamide or dimethyl sulfoxide, preferably dimethylformamide; the condensing agent described in step IIb) is selected from the group consisting of 1H-benzotriazol-1-yloxytripyrrolidine Hexafluorophosphate (PyBOP), N, N, N', N'-tetramethyl-O-(1H-benzotriazol-1-yl)urea hexafluorophosphate (HBTU) or 2-(7 Any one or a mixture of two - benzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), preferably 1H-benzotriazole-1 - oxytripyrrolidinyl hexafluorophosphate.
10. Use of the camptothecin prodrug of claim 1 for the preparation of a medicament for the treatment of cancer.

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